Enteropathogenic Escherichia coli (EPEC) strains are a leading cause of severe diarrhea worldwide. These strains use a type III secretion system (T3SS) to inject proteins into host cells, produce the attaching effacing effect, initiate the mitochondrial death pathway, and disrupt intestinal barrier function. Prior work has led to the discovery of the EspADB protein translocation apparatus and the EspF effector protein. With the overall goal of better understanding the mechanisms of EPEC pathogenesis, this proposal has four specific aims. (1) to determine the role of Abcf2 in EspF-induced apoptosis. The host Abcf2 protein is a target of EspF. Experiments will be conducted to test the hypothesis that Abcf2 is an anti-apoptotic protein that is degraded directly or indirectly by EspF. Ongoing efforts to create an abcf2 knockout mouse will be continued to study the effects of Abcf2 on apoptosis and barrier function in vivo. (2) to determine the role of EspF in alterations in barrier function. The evidence suggests that EspF is a bifunctional protein with separate effects on apoptosis that depend on mitochondrial targeting and on tight junctions that do not. The role of Abcf2 in EspF mediated disruption of barrier function will be determined and alternative pathways that lead to this effect will be identified. (3) to define interactions among translocon components EspA, EspD, and EspB. The prevailing model of the EPEC translocon proposes that the EspD and EspB proteins interact at the tip of the EspA filament that forms a pore in the host membrane through which effector molecules pass. Interactions between these proteins will be tested using genetics, biochemistry, and microscopy. (4) to determine the role of EspB and EspD in pore formation and translocation of effector proteins through the type III secretion system pore. The host membrane topology of EspB and EspD will be determined as will the ability of the purified proteins to form pores in erythrocytes and liposomes. A recessive espB allele that results in normal hemolytic activity but no effector translocation suggests a role for EspB in translocation that is separate from its role in pore formation. Whether this phenotype is due to a defect in connecting to the EspA filament or to a defect in assisting effector proteins to translocate the host membrane will be determined. The significance of this work is that it will lead to a better understanding of how bacteria inject proteins into host cells and interfere with pathways that protect the cells from death and maintain barrier function.